C-RAF Mutants that Confer Resistance to RAF Inhibitors

ABSTRACT

Nucleic acids and proteins having a mutant C-RAF sequence, and methods of identifying patients having cancer who are likely to benefit from a combination therapy and methods of treatment are provided.

RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 15/472,934,filed Mar. 29, 2017, which is a division of U.S. application Ser. No.14/387,735, filed Sep. 24, 2014, now U.S. Pat. No. 9,629,839, whichclaims the benefit under 35 U.S.C. § 371 of International ApplicationNo. PCT/US2013/029513, filed Mar. 7, 2013, which claims the benefit ofU.S. Provisional Application Nos. 61/616,999, filed Mar. 28, 2012, and61/708,372, filed Oct. 1, 2012, which are incorporated by referenceherein in their entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under federal grantnumber DP2 OD002750 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

Oncogenic mutations in the serine/threonine kinase B-RAF (also known asBRAF) are found in 50-70% of malignant melanomas. (Davies, H. et al.,Nature 417, 949-954 (2002).) Melanoma is considered to be the deadliestform of skin cancer and for the year 2012, the National Cancer Institutehas estimated 72,250 new cases which could lead to the death ofapproximately 9,180 people in the United States. Pre-clinical studieshave demonstrated that the B-RAF(V600E) mutation predicts a dependencyon the mitogen-activated protein kinase (MAPK) signaling cascade inmelanoma (Hoeflich, K. P. et al., Cancer Res. 69, 3042-3051 (2009);McDermott, U. et al., Proc. Natl Acad. Sci. USA 104, 19936-19941 (2007);Solit, D. B. et al. BRAF mutation predicts sensitivity to MEKinhibition. Nature 439, 358-362 (2006); Wan, P. T. et al., Cell 116,855-867 (2004); Wellbrock, C. et al., Cancer Res. 64, 2338-2342(2004))—an observation that has been validated by the success of RAF orMEK inhibitors in clinical trials (Flaherty, K. T. et al., N. Engl. J.Med. 363, 809-819 (2010); Infante, J. R. et al., J. Clin. Oncol. 28(suppl.), 2503 (2010); Schwartz, G. K. et al., J. Clin. Oncol. 27(suppl.), 3513 (2009).)

Recently, the FDA approved Raf inhibitor Vemurafenib (PLX4032). (Dummeret al., 2008; Infante et al., 2010; Joseph et al., 2010; Flaherty etal., 2010) However, clinical responses to targeted anticancertherapeutics are frequently confounded by de novo or acquiredresistance. (Engelman, J. A. et al., Science 316, 1039-1043 (2007);Gorre, M. E. et al., Science 293, 876-880 (2001); Heinrich, M. C. etal., J. Clin. Oncol. 24, 4764-4774 (2006); Daub, H., Specht, K. &Ullrich, A. Nature Rev. Drug Discov. 3, 1001-1010 (2004).) In theclinical and in-vitro settings, recently this phenomenon has been shownto be governed either by overexpression of a parallel signaling module(CRAF, COT) (Montagut et al., Cancer Res 68:4853-4861 (2008);Johannessen et al., Nature 468:968-972 (2010), activation of a parallelsignaling pathway (PDGFRb, IGF-1R) (Nazarian et al., Nature 468: 973-977(2010); Villanueva et al., Cancer Cell 18: 683-695 (2010), amplificationof an upstream target (BRAF) (Shi et al., Nat Commun 3:724 (2012),deletion in the target (p61BRAF) (Poulikakos et al., Nature 480:387-390(2011) or by activating mutations in the downstream target or the targetprotein itself (Mek) (Emery et al., Proc Natl Acad Sci USA 106:20411-20416 (2009); Wagle et al., JCO 29: 3085-3096 (2011). Accordingly,there remains a need for new methods for identification of resistancemechanisms in a manner that elucidates “druggable” targets for effectivelong-term treatment strategies, for new methods of identifying patientsthat are likely to benefit from the treatment strategies, and formethods of treating patients with the effective long-term treatmentstrategies.

BRIEF SUMMARY

The present invention relates to the development of resistance totherapeutic agents in the treatment of cancer and identification oftargets that confer resistance to treatment of cancer. The presentinvention also relates to identification of parallel drug targets forfacilitating an effective long-term treatment strategy and toidentifying patients that would benefit from such treatment.

In one aspect, an isolated nucleic acid molecule encoding a mutant C-RAFpolypeptide having C-RAF activity is provided. The mutant C-RAFpolypeptide includes at least one amino acid substitution as compared toa wild type C-RAF polypeptide comprising SEQ. ID. NO. 2, the at leastone amino acid substitution confers resistance to one or more RAFinhibitors on a cell expressing the mutant RAF polypeptide.

In another aspect, an expression vector in provided. The expressionvector includes the nucleic acid molecule encoding a mutant C-RAFpolypeptide having C-RAF activity where the mutant C-RAF polypeptideincludes at least one amino acid substitution as compared to a wild typeC-RAF polypeptide comprising SEQ. ID. NO. 2 and the at least one aminoacid substitution confers resistance to one or more RAF inhibitors on acell expressing the mutant RAF polypeptide.

In another aspect, a host cell is provided. The host cell includes theexpression vector.

In another aspect, an isolated mutant C-RAF polypeptide having C-RAFactivity is provided. The mutant C-RAF polypeptide includes at least oneamino acid substitution as compared to a wild type C-RAF polypeptidecomprising SEQ. ID. NO. 2 and the at least one amino acid substitutionconfers resistance to one or more RAF inhibitors on a cell expressingthe mutant C-RAF polypeptide.

In another aspect, an antibody preparation is provided. The antibodypreparation specifically binds to an isolated mutant C-RAF polypeptideof the present invention.

In yet another aspect, a method of treating a subject having cancer isprovided. The method includes extracting nucleic acid from cells of acancer of the patient and assaying at least a portion of a nucleic acidmolecule encoding a C-RAF polypeptide for the presence of one or moremutations in a nucleic acid molecule encoding a C-RAF polypeptide thatalter the identity of an amino acid residue at one or more amino acidsof the encoded C-RAF polypeptide as compared to a wild type C-RAFpolypeptide at one or more positions selected from the group consistingof 104E, 257S, 261P, 356G, 361G, 427S, 447D, 469M, 478E and 554R. Themethod also includes administering an effective amount of a RAFinhibitor and an effective amount of a second inhibitor to the subjectwhen the nucleic acid molecule includes nucleotides that alter the aminoacid residue at one or more amino acids of the encoded C-RAF polypeptideas compared to a wild type C-RAF polypeptide.

In another aspect, a method of identifying a subject having cancer whois likely to benefit from treatment with a combination therapy with aRAF inhibitor and a second inhibitor is provided. The method includesextracting nucleic acid from cells of a cancer of the patient andassaying at least a portion of a nucleic acid molecule encoding a C-RAFpolypeptide. The presence of one or more nucleotides that alter theidentity of an amino acid residue at one or more amino acids of theencoded mutant C-RAF polypeptide relative to the amino acid at one ormore positions of the wild type C-RAF polypeptide at one or more ofamino acid positions selected from the group consisting of 104E, 257S,261P, 356G, 361G, 427S, 447D, 469M, 478E and 554R indicates a need totreat the subject with a RAF inhibitor and a second inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C illustrates C-RAF mutant alleles in A375 cells (BRAFV600E)resistant to the RAF inhibitor PLX4720.

(A) The average variant score of candidate mutations across the C-RAFcoding sequence from the PLX4720 mutagenesis screen is shown. Thecorresponding amino acid substitutions from high scoring mutations (>2%)are indicated. (B) Left: crystal structure of C-RAF kinase domain(residues 340-618; grey) (PDB code: 30MV) is shown, includingrepresentative C-RAF resistance mutants (sticks) along with aspace-filling model of bound PLX4720. The DFG motif and P-loop are alsoindicated. Right: C-RAF structure rotated 90° to expose the dimerinterface residue R401 (Structures are rendered with PyMOL). (C) C-RAFdomain structure representing (CR, conserved region; RBD, Ras bindingdomain and CRD, cysteine rich domain) depicts the localization of C-RAFresistance mutants (asterisks) and three serine residues important forC-RAF regulation (circles).

FIG. 2A-G illustrates functional characterization of C-RAF resistancemutants.

(A) A375 cells expressing C-RAF (WT) and C-RAF (alleles identified) weretreated with RAF inhibitor PLX4720 in a dose dependent manner (0.08 μM,0.4 μM, 2 μM, 5 μM and 10 μM) for 90 min. Immunoblot showing pErk1/2,pMek1/2, C-RAF. α-tubulin was used as a loading control. (B) A375 cellsexpressing highly resistant C-RAF mutants were treated with 2 μM ofPLX4720 for 16 h. Shown are the levels of pMek1/2, pErk1/2, Mek, S259C-RAF, S338 C-RAF, S621 C-RAF and actin. (C) Growth inhibition curves ofA375 and C-RAF resistance alleles in response to PLX4720 and (D)vemurafenib. (E) A375 cells expressing highly resistant C-RAF mutantswere treated with 1 μM of MEK inhibitor AZD6244 for 16 h. The levels ofpMek1/2, pErk1/2, Mek, S259 C-RAF, S338 C-RAF, S621 C-RAF and actin areshown. (F) Growth inhibition curves of A375 and C-RAF resistance allelesin response to AZD6244 and (G) Mek-GSK 1120212.

FIG. 3A-B illustrates that C-RAF resistance mutants exhibit increasedassociation with B-RAF.

(A) 293/T cells expressing C-RAF resistance alleles and (B) A375 cellsexpressing C-RAF resistance alleles were immunoprecipitated with totalC-RAF. Levels of bound protein (B-RAF and 14-3-3) were assessed byimmunoblotting. Input lysate (lower panels) show 14-3-3, B-RAF, C-RAF,pMek1/2, pErk1/2, Mek, actin and S259 C-RAF, S338 C-RAF and S621 C-RAF(upper right panels). Results are representative of more than twoindependent experiments.

FIG. 4A-D illustrates the biochemical characterization of C-RAFresistance alleles using (S)-methyl1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate.

(A) Immunoblot represents pMek1/2, pErk1/2, Mek, Erk and C-RAF levels inA375 cells expressing C-RAF resistance alleles in response to 16 htreatment with 1 μM of PLX4720, (S)-methyl1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamateand AZD6244. Actin was used as a loading control. (B) Growth inhibitioncurves of A375 and C-RAF resistance alleles in response to PLX4720, (C)(S)-methyl1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamateand (D) AZD6244.

FIG. 5A-E illustrates that C-RAF resistance mutants confer resistance toVemurafenib (PLX4032).

(A) Growth inhibition curves of A375 and C-RAF resistance alleles inresponse to Vemurafenib (PLX4032). (B) C-RAF kinase activity in extractsfrom A375 cells expressing WT, S257P, P261T and G361A in the presenceand absence of Vemurafenib for 16 h. Immunoblot represents pMek1/2,pErk1/2, Mek, Erk and actin. Results are representative of threeindependent experiments. (C) Growth inhibition curves of A375 and C-RAFresistance alleles in response to PLX4720, (D) AZD6244 and (E) PLX4720and AZD6244.

FIG. 6A-D illustrates homodimerization and kinase activity of C-RAFresistance mutants.

(A) 293T cells co-expressing His/V5-tagged and Flag-tagged C-RAFresistance mutants for 48 h were immunoprecipitated with Nickel (seeMethods) to pull down His-tagged C-RAF, and Flag tagged C-RAF wasassessed by immunoblotting. Input lysate was also assessed usingantibodies that detected Flag-C-RAF, V5-C-RAF, total C-RAF, p-MEK, p-ERKand total ERK. (B) 293T cells co-expressing His/V5-tagged andFlag-tagged C-RAF resistance mutants were treated with either vehicle(DMSO) or 2 μM of vemurafenib for 1 h, and His/V5-tagged C-RAF wasimmunoprecipitated as in (A) above. Input lysates were immunoblottedusing antibodies recognizing C-RAF (S338), p-MEK, p-ERK, and actin(loading control). (C) In vitro C-Raf kinase activity was measured incell extracts derived from 293T cells transiently expressing Flag-taggedempty vector (“C”), wild type C-RAF (“WT”), and C-RAF harboring theresistance mutants S257P, P261T, G361A and E478K. Assays were performedin the presence or absence of 2 μM vemurafenib (see Methods). Inputlysate was also immunoblotted using antibodies that detect p-MEK1/2,p-ERK1/2, total MEK, total ERK and actin. (D) In vitro C-Raf kinaseactivity was measured in cell extracts derived from A375 cells(B-RAF^(V600E)) (“C”) and A375 stably expressing wild type C-RAF(“VVT”), and C-RAF harboring the resistance mutants S257P, P261T, andG361A in the presence and absence of 2 μM vemurafenib. Immunoblottingstudies were performed on input lysate using antibodies recognizingp-MEK1/2, p-ERK1/2, total MEK, total ERK, and actin. All results arerepresentative of three independent experiments.

FIG. 7A-B illustrates heterodimerization and 14-3-3 binding propertiesof C-RAF resistance mutants.

(A) 293/T cells transiently expressing the indicated C-RAF resistancemutants in the absence (−) or presence (+) of 2 μM vemurafenib for 1 hwere immunoprecipitated with C-RAF and levels of bound protein (B-RAFand 14-3-3 ζ) (upper panels) was assessed by immunoblotting. Inputlysate (lower panels) show 14-3-3 ζ, B-RAF, C-RAF, pMek1/2, pErk1/2,Mek, S338 C-RAF and actin. Results are representative of more than twoindependent experiments. (B) A375 cells stably expressing the indicatedC-RAF resistance mutants in the presence and absence of 2 μM vemurafenibfor 16 h were immunoprecipitated with C-RAF and levels of bound protein(B-RAF and 14-3-3 ζ) (upper panels) was assessed by immunoblotting.Input lysate was blotted for the same as in FIG. 7A. Results arerepresentative of more than two independent experiments.

FIG. 8A-B illustrates that C-RAF resistance mutants require dimerizationfor MEK/ERK signaling.

(A) Constructs expressing either Flag-tagged, wild-type C-RAF or theindicated C-RAF resistance mutants in the absence (left) or presence(right) of the dimerization deficient mutant R401H (pink) were expressedin 293T cells. Lysates were blotted using antibodies recognizing p-MEK,p-ERK, total MEK, or total C-RAF. (B) 293/T cells coexpressingHis-tagged C-Raf resistance mutants by themselves and in thedimerization deficient (pink) context were cultured in the absence orpresence of vemurafenib (2 μM, 1 hr). Immunoprecipitations wereperformed using Nickel beads and levels of Flag-tagged C-RAF wereassessed by immunoblotting. Input lysates blotted with antibodiesrecognizing Flag-C-RAF, V5-C-RAF, total C-RAF, p-MEK, p-ERK, S338-C-RAFand actin are also shown.

FIG. 9A-B illustrates biochemical characterization of C-RAF resistancealleles

(A) Comparison of pMek/pErk levels using A375 cells expressing C-RAFcontaining various resistance alleles that emerged from the randommutagenesis screens was expressed in A375 cells. Tubulin was included asa positive control. (B) A375 cells expressing either wild-type C-RAF orC-RAF resistance alleles were treated with the Raf inhibitor PLX4720 for90 minutes at the doses indicated. Immunoblotting studies were performedwith antibodies against p-ERK, p-MEK, and total C-RAF. Tubulin was usedas a loading control.

FIG. 10A-B illustrates homodimerization and kinase activity.

(A) 293/T cells coexpressing His/V5 tagged C-RAF resistance mutants withFlag tagged VVT-C-RAF were immunoprecipitated after 48 h with His andlevels of Flag tagged C-RAF interaction was assessed by immunoblotting.Input lysate represents Flag-C-RAF, V5-C-RAF, C-RAF, pMek, pErk and Erk.(B) 293/T cells were transfected with the dimerization deficient C-RAFmutant R401H and gatekeeper mutant T421N and Mek/Erk sensitivity wasdetected in the presence of vemurafenib (2 μM) for 1 h. T421N was usedas a negative control and G361A was used as a positive control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the development of resistance totherapeutic agents in the treatment of cancer and identification oftargets that confer resistance to treatment of cancer. The presentinvention also relates to identification of parallel drug targets forfacilitating an effective long-term treatment strategy and toidentifying patients that would benefit from such treatment.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, immunology,microbiology, cell biology and recombinant DNA, which are within theskill of the art. See e.g., Sambrook, Fritsch and Maniatis, MOLECULARCLONING: A LABORATORY MANUAL, (Current Edition); CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel et al. eds., (Current Edition)); theseries METHODS IN ENZYMOLOGY (Academic Press, Inc.); PCR 2: A PRACTICALAPPROACH (Current Edition); ANTIBODIES, A LABORATORY MANUAL and ANIMALCELL CULTURE (R. I. Freshney, ed. (1987)). DNA Cloning: A PracticalApproach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N.Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames & S.Higgins, eds., Current Edition); Transcription and Translation (B. Hames& S. Higgins, eds., Current Edition); Fundamental Virology, 2nd Edition,vol. I & II (B. N. Fields and D. M. Knipe, eds.)

The mitogen-activated protein kinase (MAPK) cascade is a criticalintracellular signaling pathway that regulates signal transduction inresponse to diverse extracellular stimuli, including growth factors,cytokines, and proto-oncogenes. Activation of this pathway results intranscription factor activation and alterations in gene expression,which ultimately lead to changes in cellular functions including cellproliferation, cell cycle regulation, cell survival, angiogenesis andcell migration. Classical MAPK signaling is initiated by receptortyrosine kinases at the cell surface, however many other cell surfacemolecules are capable of activating the MAPK cascade, includingintegrins, heterotrimeric G-proteins, and cytokine receptors.

Ligand binding to a cell surface receptor, e.g., a receptor tyrosinekinase, typically results in phosphorylation of the receptor. Theadaptor protein Grb2 associates with the phosphorylated intracellulardomain of the activated receptor, and this association recruits guaninenucleotide exchange factors including SOS-I and CDC25 to the cellmembrane. These guanine nucleotide exchange factors interact with andactivate the GTPase Ras. Common Ras isoforms include K-Ras, N-Ras, H-Rasand others. Following Ras activation, the serine/threonine kinase Raf(e.g., A-Raf, B-Raf, C-Raf or Raf-1) is recruited to the cell membranethrough interaction with Ras or in a Ras independent manner in thecytosol where it undergoes conformational changes and binding toscaffold proteins such as 14-3-3 (King et al., Nature 396; 180-183(1998); Chaudhary et al., Curr Biol 10: 551-554 (2000); Avruch et al.,Endo Rev 56: 127-156 (2001), Wellbrock et al., Nat Rev Mol Cell Biol 5:875-885 (2004). 14-3-3 binding and stabilization/activation of CRAF isgoverned by phosphorylation of activating residues such as S338, Y341 inthe negative charge regulatory region (N-region) and S621 in theC-terminus, outside the kinase domain and dephosphorylation of negativeregulatory residues such as S259 in the CR2 domain (FIG. 1C) andnumerous other phosphorylation sites distributed throughout the proteinwhich further reflects its complex regulation (Avruch et al., Id.(2001); Wellbrock et al., Id. (2004); Garnett et al., Mol. Cell 20:963-969 (2005). CRAF activation is also induced by artificial homodimerformation (Avruch et al., Id. (2001); Wellbrock et al., Id., (2004).)

Raf is then phosphorylated. Raf directly activates MEKI and MEK2 byphosphorylation of two serine residues at positions 217 and 221.Following activation, MEKI and MEK2 phosphorylate tyrosine (Tyr-185) andthreonine (Thr-183) residues in serine/threonine kinases ErkI and Erk2,resulting in Erk activation. Activated Erk regulates many targets in thecytosol and also translocates to the nucleus, where it phosphorylates anumber of transcription factors regulating gene expression. Erk kinasehas numerous targets, including Elk-I, c-EtsI, c-Ets2, p90RSKI, MNKI,MNK2, MSKI, MSK2 and TOB. While the foregoing pathway is a classicalrepresentation of MAPK signaling, there is considerable cross talkbetween the MAPK pathway and other signaling cascades.

Aberrations in MAPK signaling have a significant role in cancer biology.Altered expression of Ras is common in many cancers, and activatingmutations in Ras have also been identified. Such mutations are found inup to 30% of all cancers, and are especially common in pancreatic (90%)and colon (50%) carcinomas. In addition, activating B-Raf mutations havebeen identified in melanoma and ovarian cancer. The most commonmutation, BRAF^(V600E), results in constitutive activation of thedownstream MAP kinase pathway and is required for melanoma cellproliferation, soft agar growth, and tumor xenograft formation. CRAFamplification have been implicated in prostate cancer and bladder cancer(Edwards et al., 2003; Simon et al., 2001), besides chromosomaltranslocations in stomach cancer and pilocytic astrocytomas (Shimizu etal., 1986; Jones et al., 2009). However, the occurrence rate of CRAFmutations in human cancers is 1% (COSMIC) which is attributable to itslow basal kinase activity when compared to BRAF (Marais et al., Science280: 109-112 (1997); Emuss et al., Cancer Res 65: 9719-9726 (2005);Garnett et al., Mol. Cell 20: 963-969 (2005)). Based on the defined roleof MAPK over-activation in human cancers, targeting components of theMAPK pathway with specific inhibitors is a promising approach to cancertherapy. However, patients may have innate resistance or acquireresistance to these promising therapies. Identification of resistanceconferring mutations in target kinases, diagnostic and/or prognosticmarkers and treatment therapies for these patients with innate oracquired resistance are described below.

C-Raf Mutations

While treatment of cancer with RAF inhibitors, such as PLX4032, is apromising therapeutic approach, patients receiving such therapiesfrequently relapse or fail to respond, and as a result the patients'disease progresses. As described herein, the present invention relatesto the discovery of mutations in C-RAF that confer resistance to RAFinhibitors, some of which are currently in clinical development.Acquisition of such a mutation in cancer cells makes cells of thepatient resistant to treatment with certain RAF inhibitors. In exemplaryembodiments, the invention regards development of resistance to RAFinhibitors, that may include but are not limited to RAF265, sorafenib,SB590885, PLX 4720, PLX4032, GDC-0879, ZM 336372 and (S)-methyl1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate.By way of non-limiting example, exemplary RAF inhibitors are shown inTable 1. The RAF inhibitor (S)-methyl1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamateis generically and specifically covered (compound 9, table 1, page 50 ofthe provisional; Compound 9 on pg. 72 of PCT publication WO2011/025927.

The clinical emergence of a C-RAF mutation conferring resistance to aRAF inhibitor as described herein suggests that the biological relevanceof RAF/MEK-associated dependency is maintained even in advanced stagesof malignancy. Thus, the failure of RAF inhibitors to elicit durabletumor responses in many malignancies, including melanomas may indicatesuboptimal drug potency or pharmacodynamics in the clinical setting.Based on the findings described herein, treatment modalities involvingtargeted agents in RAF- or MEK-driven tumors may benefit from morepotent drugs, altered dosing of existing drugs, or combined RAF and MEKinhibition. Exemplary RAF inhibitors include, but are not limited to theinhibitors listed in Table I. Non-limiting examples of MEK inhibitorsinclude, AZD6244; CI-1040; PD184352; PD318088, PD98059, PD334581,RDEA119,6-Methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)-quinoline-3-carbonitrileand4-[3-Chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)-phenylamino]-6-methoxy-7-(3-morpholin-4-yl-propoxy)-quinoline-3-carbonitrile.Exemplary MEK inhibitors are shown in Table 2. These therapeuticinnovations, together with robust tumor genomic profiling to stratifypatients, should speed the advent of personalized cancer treatment incancers with “druggable” oncogene mutations.

TABLE 1 Exemplary RAF Inhibitors Name CAS No. Structure 1 RAF265 927880-90-

2 Sorafenib Tosylate Nexavar Bay 43-9006 475207- 59-1

Sorafenib 4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methyl-pyridine-2-carboxamide 284461- 73-0

3 SB590885

4 PLX4720 918505- 84-7

5 PLX4032 1029872- 54-5

6 GDC-0879 905281- 76-7

7 ZM 336372 208260- 29-1

8 (S)-methyl 1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2- ylcarbamate

TABLE 2 Exemplary MEK Inhibitors Name CAS No. Structure 1Cl-1040/PD184352 212631-79-3

2 AZD6244 606143-52-6

3 PD318088 391210-00-7

4 PD98059 167869-21-8

5 PD334581

6 RDEA119 N-[3,4-difluoro-2-[(2- fluoro-4- iodophenyl)amino]-6-methoxyphenyl]-1-[(2R)- 2,3-dihydroxypropyI]- Cyclopropanesulfonamide923032-38-6

7 6-Methoxy-7-(3- morpholin-4-yl-propoxy)- 4-(4-phenoxy-phenylamino)-quinoline- 3-carbonitrile

8 4-[3-Chloro-4-(1-methyl- 1H-imidazol-2- ylsulfanyl)-phenylamino]-6-methoxy-7-(3- morpholin-4-yl-propoxy)- quinoline-3-carbonitrile

In various embodiments, the present invention relates to methods ofidentifying mutations in a C-RAF polypeptide, or mutations in a nucleicacid molecule encoding the C-RAF polypeptide, that confer resistance oncells expressing the C-RAF polypeptide to drugs that inhibit RAFactivity. A “mutant C-RAF polypeptide,” as referenced herein, includes aC-RAF polypeptide including one or more mutations that confer resistanceto one or more known RAF inhibitors. Likewise, a “mutant C-RAF nucleicacid molecule,” as referenced herein, includes a nucleic acid moleculethat encodes a mutant C-RAF polypeptide. Nucleic acid molecules encodingC-RAF polypeptides that include one or more mutations can be createdusing any suitable method known in the art, including, for example,random mutagenesis or site-directed mutagenesis of a wild-type C-RAFnucleic acid sequence, which can be conducted in E. coli. In exemplaryembodiments, the wild-type C-RAF nucleic acid sequence is a humanwild-type MEK1 nucleic acid sequence. In specific embodiments, thewild-type C-RAF nucleic acid sequence is wild-type human C-RAF (SEQ IDNO: 1) (Accession Number BC018119.2.). The mutant C-RAF nucleic acidmolecules can then be screened in cells otherwise sensitive to treatmentwith a RAF inhibitor to identify a nucleic acid that encodes a mutantC-RAF polypeptide compared to a wild-type C-RAF polypeptide that isresistant to treatment with the RAF inhibitor. In some embodiments, theC-RAF polypeptide is the wild-type human C-RAF (SEQ ID NO: 2)(Swiss-Prot ID # is P04049-10).

Any suitable method can be used to screen mutant C-RAF nucleic acids andmutant C-RAF polypeptides for resistance to treatment with a RAFinhibitor. For example, a nucleic acid molecule encoding a mutant C-RAFpolypeptide can be expressed in cells otherwise sensitive to treatmentwith a RAF. An exemplary cell line useful for this purpose is themelanoma cell line A375. Following expression of the mutant C-RAFpolypeptide, the cells can be treated with a RAF. The activity of themutant C-RAF polypeptide can then be measured and compared to theactivity of a wild-type C-RAF polypeptide similarly expressed andtreated with the RAF inhibitor. Activity of a C-RAF polypeptide can bedetermined by, for example, measuring proliferation or viability ofcells following treatment with the RAF inhibitor, wherein proliferationor viability are positively correlated with C-RAF activity. Cell growth,proliferation, or viability can be determined using any suitable methodknown in the art. In one embodiment, cell growth can be determined usingwell-based cell proliferation/viability assays such as MTS or Cell TiterGLo, in which cell growth in the presence of a RAF inhibitor isexpressed as a percentage of that observed in untreated cells culturedin the absence of the RAF inhibitor. In certain embodiments, resistanceis defined as a shift in the G150 value of at least 2 fold, morepreferably at least 3 fold, most preferably at least 4-5 fold, withrespect to a suitable control. In other embodiments, resistance isdefined as a GI50 value of ˜1 uM). Activity of a C-RAF polypeptide canalso be measured by, for example, determining the relative amount ofphosphorylated ERK1/2 present in the cell following treatment with theRAF inhibitor. Activity of a wild-type or mutant C-RAF polypeptide canalso be determined using an in vitro phosphorylation assay, in whichMEK1 activity is determined by measuring the proportion ofphosphorylated ERK 1/2 substrate in the assay following treatment withthe RAF or MEK inhibitor. A mutant C-RAF polypeptide having greateractivity than a wild-type C-RAF polypeptide following treatment with aRAF inhibitor is identified as containing a mutation that confersresistance to a RAF inhibitor. The mutation conferring resistance to aRAF inhibitor can then be identified by sequencing the nucleic acidencoding the mutant C-RAF polypeptide, or by sequencing the mutant C-RAFpolypeptide directly.

In this manner, as well as using massively parallel sequence methods, asdescribed in Example 1, amino acid substitutions were identified in theC-RAF polypeptide that when mutated confer resistance to the RAFinhibitors PLX4032, PLX4720 and (S)-methyl1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate.In particular, substitutions at one or more of the following amino acidsof the human C-RAF polypeptide confer resistance to RAF inhibitorsincluding 104E, 257S, 261P, 356G, 361G, 427S, 447D, 469M, 478E and 554R.In certain embodiments, the mutant C-RAF polypeptide includes a mutationwith respect to the wild-type human C-RAF polypeptide at one or more ofthese amino acid residues. In exemplary embodiments, the mutant C-RAFpolypeptide includes one or more of the following resistance mutations:104E>K, 257S>P, 261P>T, 356G>E, 361G>A, 427S>T, 447D>N, 469M>I, 478E>Kand 554R>K.

Isolated Nucleic Acid Molecules

The present invention concerns polynucleotides or nucleic acid moleculesrelating to the C-RAF gene and its respective gene product. Thesepolynucleotides or nucleic acid molecules are isolatable and purifiablefrom mammalian cells. In particular aspects of the invention, theisolated C-RAF nucleic acid molecules described herein comprise amutation conferring resistance to one or more RAF inhibitors. A “mutantC-RAF nucleic acid molecule,” as referenced herein, includes a C-RAFnucleic acid molecule that encodes a mutant C-RAF polypeptide, i.e., aC-RAF polypeptide including one or more mutations that confer resistanceto one or more RAF inhibitors.

It is contemplated that an isolated and purified C-RAF nucleic acidmolecule, e.g., a mutant C-RAF nucleic acid molecule, can take the formof RNA or DNA. As used herein, the term “RNA transcript” refers to anRNA molecule that is the product of transcription from a DNA nucleicacid molecule. Such a transcript can encode for one or more proteins.

As used in this application, the term “polynucleotide” refers to anucleic acid molecule, RNA or DNA, that has been isolated, such as beingfree of total genomic nucleic acid. Therefore, a “polynucleotideencoding C-RAF” refers to a nucleic acid segment that includes C-RAFcoding sequences, yet is isolated away from, or purified and free of,total genomic DNA and proteins. When the present application refers tothe function or activity of a C-RAF-encoding polynucleotide or nucleicacid, it is meant that the polynucleotide encodes a molecule that iscapable of performing an activity of a wild-type C-RAF polypeptide, forexample, phosphorylation of the ERK1/2 substrate.

The term “cDNA” is intended to refer to DNA prepared using RNA as atemplate. It also is contemplated that a given C-RAF-encoding nucleicacid or C-RAF gene from a given cell may be represented by naturalvariants or strains that have slightly different nucleic acid sequencesbut, nonetheless, encode an active C-RAF polypeptide. In a preferredembodiment, the active C-RAF polypeptide is an active human C-RAFpolypeptide. In particularly preferred embodiments, the active C-RAFpolypeptide is a mutant C-RAF polypeptide that has an activity of awild-type C-RAF polypeptide, but which is resistant to one or more knownRAF inhibitors. Consequently, certain aspects of the present inventionencompass derivatives of C-RAF nucleic acids or polypeptides withminimal nucleic acid or amino acid changes, but that possess the samebiological function.

In some embodiments, the invention relates to recombinant vectorsincorporating DNA sequences that encode mutant C-RAF polypeptides orpeptides that include within its amino acid sequence a contiguous aminoacid sequence in accordance with, or essentially corresponding to mutantC-RAF polypeptides. In exemplary embodiments, the invention relates toisolated DNA segments and recombinant vectors incorporating DNAsequences that encode a C-RAF polypeptide that includes within its aminoacid sequence a contiguous amino acid sequence of a C-RAF polypeptidecomprising one or more mutations that confer resistance to one or moreRAF inhibitors.

The nucleic acid segments used in the present invention, regardless ofthe length of the coding sequence itself, can be combined with other DNAor RNA sequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length can vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength can be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol. A “heterologous” sequence refers to a sequence that is foreignor exogenous to the remaining sequence. A heterologous gene refers to agene that is not found in nature adjacent to the sequences with which itis now placed.

In some embodiments, the nucleic acid sequence may encode a mutant C-RAFpolypeptide having C-RAF activity where at least one amino acidsubstitution occurs at one or more amino acid positions including thefollowing: 104E, 257S, 261P, 356G, 361G, 427S, 447D, 469M, 478E and554R. In other embodiments, the mutant C-RAF polypeptide includes one ormore of the following resistance mutations: 104E>K, 257S>P, 261P>T,356G>E, 361G>A, 427S>T, 447D>N, 469M>I, 478E>K and 554R>K.

Expression Vectors and Host Cells

The present invention encompasses expression vector compositions and theuse of such vectors to encode for a C-RAF polypeptide, e.g., a mutantC-RAF polypeptide, as well as host cell compositions into which suchexpression vectors have been introduced. The term “vector” is used torefer to a carrier nucleic acid molecule into which a nucleic acidsequence can be inserted for introduction into a cell where it can bereplicated. A nucleic acid sequence can be “exogenous,” which means thatit is foreign to the cell into which the vector is being introduced orthat the sequence is homologous to a sequence in the cell but in aposition within the host cell nucleic acid in which the sequence isordinarily not found. Vectors include plasmids, cosmids, viruses(bacteriophage, animal viruses, and plant viruses), and artificialchromosomes (e.g., YACs). One of skill in the art would be well equippedto construct a vector through standard recombinant techniques.

The term “expression vector” or “expression construct” refers to avector containing a nucleic acid sequence coding for at least part of agene product capable of being transcribed. In some cases, RNA moleculesare then translated into a protein, protein, or peptide. In other cases,these sequences are not translated, for example, in the production ofantisense molecules or ribozymes. Expression vectors can contain avariety of “control sequences,” which refer to nucleic acid sequencesnecessary for the transcription and possibly translation of an operablylinked coding sequence in a particular host organism. In addition tocontrol sequences that govern transcription and translation, vectors andexpression vectors can contain nucleic acid sequences that serve otherfunctions as well.

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. A cell comprising a C-RAF polynucleotide, eithermutated or wild-type, can be employed in the invention. All of theseterms also include their progeny, which refers to any and all subsequentgenerations. It is understood that all progeny may not be identical dueto deliberate or inadvertent mutations. In the context of expressing aheterologous nucleic acid sequence, “host cell” refers to a prokaryoticor eukaryotic cell, and it includes any transformable organisms that iscapable of replicating a vector and/or expressing a heterologous geneencoded by a vector. A host cell can, and has been, used as a recipientfor vectors. A host cell may be “transfected” or “transformed,” whichrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A transformed cell includes the primarysubject cell and its progeny. A “recombinant host cell” refers to a hostcell that carries a recombinant nucleic acid, i.e. a nucleic acid thathas been manipulated in vitro or that is a replicated copy of a nucleicacid that has been so manipulated. A host cell can be derived fromprokaryotes or eukaryotes, depending upon whether the desired result isreplication of the vector, expression of part or all of thevector-encoded nucleic acid sequences, or production of infectious viralparticles.

Isolated Polypeptide Molecules

Another aspect of the invention pertains to isolated and/or purifiedC-RAF polypeptides, and biologically active portions thereof. Inparticular aspects of the invention, the C-RAF polypeptides describedherein comprise a mutation at one or more amino acids conferringresistance to one or more RAF inhibitors. A “mutant C-RAF polypeptide”,as referenced herein, includes a C-RAF polypeptide including a mutationat one or more amino acids positions that confer resistance to one ormore RAF inhibitors.

Biologically active portions of a C-RAF polypeptide include peptidescomprising amino acid sequences derived from the amino acid sequence ofa C-RAF polypeptide, e.g., the amino acid sequence shown in SEQ ID NO:2, which include fewer amino acids than a full length C-RAF polypeptide,and exhibit at least one activity of a C-RAF polypeptide. Typically,biologically active portions (peptides, e.g., peptides which are, forexample, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or moreamino acids in length) comprise a domain or motif with at least oneactivity of a C-RAF polypeptide. Moreover, other biologically activeportions, in which other regions of the protein are deleted, can beprepared by recombinant techniques and evaluated for one or more of theactivities described herein. Preferably, the biologically activeportions of a C-RAF polypeptide include one or more selecteddomains/motifs or portions thereof having biological activity.

C-RAF polypeptides may be produced by recombinant DNA techniques. Forexample, a nucleic acid molecule encoding the protein is cloned into anexpression vector (as described above), the expression vector isintroduced into a host cell (as described above) and the C-RAFpolypeptide is expressed in the host cell. The C-RAF polypeptide canthen be isolated from the cells by an appropriate purification schemeusing standard protein purification techniques. Alternative torecombinant expression, a C-RAF polypeptide can be synthesizedchemically using standard peptide synthesis techniques. Moreover, anative C-RAF polypeptide and/or a mutant C-RAF polypeptide can beisolated from cells (e.g., cancer cells), for example using ananti-C-RAF antibody, which can be produced by standard techniquesutilizing a C-RAF polypeptide or fragment thereof of this invention.

C-RAF chimeric or fusion proteins may also be used. As used herein, aMEK1 “chimeric protein” or “fusion protein” comprises a C-RAFpolypeptide operatively linked to a non-C-RAF polypeptide. A “C-RAFpolypeptide” refers to a protein having an amino acid sequencecorresponding to a C-RAF polypeptide, whereas a “non-C-RAF polypeptide”refers to a protein having an amino acid sequence corresponding to aprotein which is not substantially homologous to the C-RAF polypeptide,e.g., a protein which is substantially different from the C-RAFpolypeptide, which does not display a C-RAF activity and which isderived from the same or a different organism. Within the fusionprotein, the term “operatively linked” is intended to indicate that theC-RAF polypeptide and the non-C-RAF polypeptide are fused in-frame toeach other. The non-C-RAF polypeptide can be fused to the N-terminus orC-terminus of the C-RAF polypeptide. For example, in one embodiment thefusion protein is a GST-C-RAF fusion protein in which the C-RAF aminoacids are fused to the C-terminus of the GST polypeptide. Such fusionproteins can facilitate the purification of recombinant C-RAFpolypeptide. In another embodiment, the fusion protein is a C-RAFpolypeptide containing a heterologous signal sequence at its N-terminus.In certain host cells (e.g., mammalian host cells), expression and/orsecretion of a MEK1 protein can be increased through use of aheterologous signal sequence.

Mutant C-RAF polypeptide can be generated by mutagenesis of a wild-typeC-RAF polypeptide, or of the nucleic acid molecule encoding a wild-typeC-RAF polypeptide. Mutant C-RAF polypeptide can also be identified byscreening combinatorial libraries of C-RAF mutants for a mutant C-RAFpolypeptide having a desired activity, e.g., resistance to one or moreRAF inhibitors. Several techniques are known in the art for screeninggene products of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having aselected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by combinatorial mutagenesis. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected.

Antibodies

The polypeptides expressed from the polynucleotides of the invention canbe used for generating antibodies. In some embodiments, the antibodiescan be used to detect and quantitate expression of the mutant C-RAFpolypeptides. In some embodiments, the antibodies can be used to alterthe activity of a mutant C-RAF polypeptide. Polypeptides expressed fromthe polynucleotides of the invention comprising at least six, eight,ten, twelve, fifteen, twenty or thirty consecutive amino acids can beused as immunogens. The polypeptides can be used to obtain a preparationof antibodies which specifically bind to a mutant C-Raf polypeptide ofthe invention having one or more amino acid substitutions at one or moreof the following amino acids of the human C-RAF polypeptide that conferresistance to RAF inhibitors including 104E, 257S, 261P, 356G, 361G,427S, 447D, 469M, 478E and 554R. In exemplary embodiments, the mutantC-RAF polypeptide includes one or more of the following resistancemutations: 104E>K, 257S>P, 261P>T, 356G>E, 361G>A, 427S>T, 447D>N,469M>, 478E>K and 554R>K.

The antibodies can be monoclonal and polyclonal antibodies, single chainantibodies, chimeric antibodies, bifunctional/bispecific antibodies,humanized antibodies, human antibodies, and complementary determiningregion (CDR)-grafted antibodies, that are specific for the targetprotein or fragments thereof; and also include antibody fragments,including Fab, Fab′, F(ab′)2, scFv, Fv, camelbodies, or microantibodies.An antibody can also refer to an anti-idiotype antibody, i.e., anantibody directed against the antigen specific part of the sequence ofan antibody and thus recognizes the binding sites of other antibodies;or an anti-anti-idiotype antibody, i.e., an antibody with a combiningsite that mimics the epitope on the antigen that was used to generatethe original antibody. Techniques for raising antibodies are well knownin the art.

Single chain antibodies can also be constructed. Single chain antibodieswhich specifically bind to a polypeptide expressed from thepolynucleotides of the invention can be isolated, for example, fromsingle-chain immunoglobulin display libraries, as are known in the art.The library is “panned” against a polypeptide, and a number of singlechain antibodies which bind different epitopes of the polypeptide withhigh-affinity can be isolated. Hayashi et al., 1995, Gene 160: 129-30.Such libraries are known and available to those in the art. Theantibodies can also be constructed using the polymerase chain reaction(PCR), using hybridoma cDNA as a template. Thirion et al., 1996, Eur. J.Cancer Prey. 5: 507-11.

The single chain antibody can be mono- or bi-specific, and can bebivalent or tetravalent. Construction of tetravalent bispecific singlechain antibodies is taught in Coloma and Morrison, 1997, Nat.Biotechnol. 15: 159-63 Construction of bivalent bispecific single chainantibodies is taught in Mallender and Voss, 1994, J. Biol. Chem. 269:199-206.

A nucleotide sequence encoding the single chain antibody can then beconstructed using manual or automated nucleotide synthesis, cloned intoDNA expression vectors using standard recombinant DNA methodologies, andintroduced into cells which express the selected gene, as describedbelow. Alternatively, the antibodies can be produced directly usingfilamentous phage technology Verhaar et al., 1995, Int. J. Cancer61:497-501; Nicholls et al., 1993. J. Immunol. Meth. 165:81-91.

The antibodies bind specifically to the epitopes of the polypeptidesexpressed from the polynucleotides of the invention. In a preferredembodiment, the epitopes are not present on other human proteins.Typically a minimum number of contiguous amino acids to encode anepitope is 6, 8, or 10. However, more can be used, for example, at least15, 25, or 50, especially to form epitopes which involve non-contiguousresidues or particular conformations.

Antibodies that bind specifically to the polypeptides include those thatbind to full-length polypeptides. Specific binding antibodies do notdetect other proteins on Western blots of human cells, or provide asignal at least ten-fold lower than the signal provided by the targetprotein of the invention. Antibodies which have such specificity can beobtained by routine screening. In a preferred embodiment of theinvention, the antibodies immunoprecipitate the polypeptides expressedfrom the polynucleotides of the invention from cell extracts orsolution. Additionally, the antibodies can react with polypeptidesexpressed from the polynucleotides of the invention in tissue sectionsor on Western blots of polyacrylamide gels. Preferably the antibodies donot exhibit nonspecific cross-reactivity with other human proteins onWestern blots or in immunocytochemical assays.

Techniques for purifying antibodies to the polypeptides expressed fromthe polynucleotides of the invention are available in the art. In apreferred embodiment, the antibodies are passed over a column to which aparticular protein or polypeptide expressed from the polynucleotides ofthe invention is bound. The bound antibodies are then eluted, forexample, with a buffer having a high salt concentration.

Detection of Mutations

In another aspect, the invention pertains to methods of detecting thepresence of a mutant C-RAF polypeptide in a sample (e.g., a biologicalsample from a cancer patient). A variety of screening methods can beused to detect the presence of a mutant C-RAF polypeptide of theinvention in a sample, e.g., a nucleic acid and/or a protein sample. Inspecific embodiments, the sample includes a cell or cell extract. Inexemplary embodiments, the sample is obtained from a subject, e.g., asubject having cancer.

Methods for detecting the presence of resistance mutations in genomicDNA, cDNA, and RNA (i.e., mRNA) containing a sequence encoding a C-RAFpolypeptide, or biologically active portion thereof, can be used withinthe scope of the present invention. Likewise, methods for detecting thepresence of resistance mutations in C-RAF polypeptide, or biologicallyactive portions thereof, can be used within the scope of the presentinvention. In particular embodiments, methods including, but not limitedto, the following can be used to detect the presence of a C-RAFpolypeptide, or a nucleic acid molecule encoding C-RAF polypeptide,having a mutation at one or more amino acid positions as compared to thewild-type C-RAF polypeptide (SEQ ID NO: 2). In some embodiments,antibodies directed to a mutant C-RAF polypeptide may be used to detectthe presence of the mutant polypeptide.

Point mutations can be detected using any suitable method known in theart, including, for example, denaturing gradient gel electrophoresis(“DGGE”), restriction fragment length polymorphism analysis (“RFLP”),chemical or enzymatic cleavage methods, direct sequencing of targetregions amplified by PCR (see above), single-strand conformationpolymorphism analysis (“SSCP”), polymerase chain reaction, sequencing,hybridization, or “hybrid capture” followed by pyrosequencing orsingle-molecule sequencing. Other methods for detecting mutations knownto one skilled in the art may also be used.

Screening methods can be performed to screen an individual for theoccurrence of the mutations identified above. For example, in oneembodiment, a sample (such as blood or other bodily fluid or cell ortissue sample) is taken from a patient for analysis. In an exemplaryembodiment, the patient is a cancer patient. Methods suitable forprocessing such samples for detection of a mutation in a C-RAF nucleicacid or a C-RAF polypeptide are known in the art, and the skilledartisan may adapt the processing of such samples in accordance with thechosen method of detection.

The presence or absence of one or more mutations described hereindetermines the likelihood of the screened individuals to resist therapywith a RAF inhibitor. According to methods provided by the invention,these results will be used to adjust and/or alter the dose of the RAFinhibitor, or to select a course of treatment using a second inhibitor.In some embodiments, the second inhibitor may be a MEK inhibitor.Effective treatment of a subject having cancer can comprise theeradication of a cancer cell, the cessation or reduction of cancer (suchas solid tumor) growth rate, or the amelioration of at least one cancersymptom.

The resistance mutations in C-RAF polypeptide, or in nucleic acidmolecules encoding C-RAF polypeptide, can be detected using any suitablemethods known in the art, or modifications thereof, including themethods described below. Such methods include the use of allele-specificpolymerase chain reaction, direct or indirect sequencing of the site,the use of restriction enzymes where the respective alleles of the sitecreate or destroy a restriction site, the use of allele-specifichybridization probes, the use of antibodies that are specific for mutantC-RAF polypeptide, or any other biochemical interpretation.

Diagnostic/Prognostic Markers for Resistance to Targeted Therapies

In some aspects, the present invention relates to methods of detectingthe presence of one or more diagnostic or prognostic markers in a sample(e.g. a biological sample from a cancer patient). A variety of screeningmethods known to one of skill in the art may be used to detect thepresence of the marker in the sample including DNA, RNA and proteindetection. The techniques can be used to determine the presence orabsence of a mutation in a sample obtained from a patient. In someembodiments, the patient may have innate or acquired resistance tokinase targeted therapies, including B-RAF inhibitors or pan-RAFinhibitors. For example, the patient may have an innate or acquiredresistance to RAF inhibitors PLX4720 and/or PLX4032 and/or (S)-methyl1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate.In one embodiment, identification of a C-RAF nucleic acid or polypeptideincluding one or more mutations described herein in a cancer-cellcontaining sample obtained from a patient indicates that the patient isat a relatively high risk of relapse or lack of response to treatmentwith a RAF inhibitor. Identification of one or more C-RAF mutationsdescribed above in a patient assists the physician in determining andimplementing a treatment protocol for the patient. For example, in apatient having one or more mutations in the C-RAF polypeptide identifiedabove, the physician may treat the patient with a combination therapy asdescribed in more detail below.

Identification of resistance mutations in the C-RAF polypeptide alsoallows for the screening of patients having a cancer in order todetermine the presence or absence of a C-RAF resistance mutation at oneor more amino acid positions in the cancer. Determining the presence orabsence of one or more C-RAF resistance mutations in a cancer allows foralteration of the treatment strategy of a cancer patient. Suchalterations can include, for example, starting or stopping treatmentwith a RAF inhibitor or a MEK inhibitor, giving a combination therapy,providing sequential dosing of a RAF inhibitor and a second inhibitorand the like.

In some embodiments, the RAF resistance mutations may be identified in anucleic acid encoding a mutant C-RAF polypeptide having C-RAF activity,where the mutant C-RAF polypeptide includes at least one amino acidsubstitution as compared to a wild type C-RAF polypeptide shown in SEQID NO: 2 and where the at least one amino acid substitution confersresistance to one or more RAF inhibitors on a cell expressing the mutantRAF polypeptide. In some embodiments, the RAF resistance mutations maybe identified in a mutant C-RAF polypeptide having C-RAF activity, wherethe mutant C-RAF polypeptide includes at least one amino acidsubstitution as compared to a wild type C-RAF polypeptide shown in SEQ.ID. NO. 2, and where the at least one amino acid substitution confersresistance to one or more RAF inhibitors on a cell expressing the mutantC-RAF polypeptide. In some embodiments, the substitution at one or moreof the following amino acids of the wild-type C-RAF polypeptide conferresistance to RAF inhibitors including 104E, 257S, 261P, 356G, 361G,427S, 447D, 469M, 478E and 554R. In some embodiments, the substitutionof one or more amino acids of the wild-type C-RAF polypeptide isselected from the group consisting of 104E>K, 257S>P, 261P>T, 356G>E,361G>A, 427S>T, 447D>N, 469M>I, 478E>K and 554R>K. In some embodiments,the substitution of one or more amino acids of the wild-type C-RAFpolypeptide is selected from the group consisting of 257S, 261P and361G.

Methods of Treatment

In various embodiments, the invention provides methods for treatment ofa patient having cancer. The methods generally comprise administrationof a first inhibitor and a second inhibitor. One inhibitor may be a RAFinhibitor. Exemplary RAF inhibitors are shown in Table 1 above. Oneinhibitor may be a MEK inhibitor (see Table 2 illustrating exemplary MEKinhibitors). In some embodiments, a combination therapy for cancer isprovided, comprising an effective amount of a RAF inhibitor and aneffective amount of a second inhibitor. In some embodiments the secondinhibitor is a MEK inhibitor.

In exemplary embodiments of the foregoing aspects, the RAF inhibitorprovided herein is PLX4720, PLX4032, BAY 43-9006 (Sorafenib), ZM 336372,RAF 265, AAL-881, LBT-613, (S)-methyl1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamateor CJS352. Additional exemplary RAF inhibitors useful for combinationtherapy include pan-RAF inhibitors, inhibitors of B-RAF, inhibitors ofA-RAF, and inhibitors of RAF-1. Additional RAF inhibitors known in theart may also be used.

As a non-limiting example, the MEK inhibitor provided herein can beCI-1040, AZD6244, PD318088, PD98059, PD334581, RDEA119,6-Methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)-quinoline-3-carbonitrileor4-[3-Chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)-phenylamino]-6-methoxy-7-(3-morpholin-4-yl-propoxy)-quinoline-3-carbonitrile,Roche compound RG7420, or combinations thereof. Additional MEKinhibitors known in the art may also be used.

Administration of the combination includes administration of thecombination in a single formulation or unit dosage form, administrationof the individual agents of the combination concurrently but separately,or administration of the individual agents of the combinationsequentially by any suitable route. The dosage of the individual agentsof the combination may require more frequent administration of one ofthe agents as compared to the other agent in the combination. Therefore,to permit appropriate dosing, packaged pharmaceutical products maycontain one or more dosage forms that contain the combination of agents,and one or more dosage forms that contain one of the combinations ofagents, but not the other agent(s) of the combination.

Agents may contain one or more asymmetric elements such as stereogeniccenters or stereogenic axes, e.g., asymmetric carbon atoms, so that thecompounds can exist in different stereoisomeric forms. These compoundscan be, for example, racemates or optically active forms. For compoundswith two or more asymmetric elements, these compounds can additionallybe mixtures of diastereomers. For compounds having asymmetric centers,it should be understood that all of the optical isomers and mixturesthereof are encompassed. In addition, compounds with carbon-carbondouble bonds may occur in Z- and E-forms; all isomeric forms of thecompounds are included in the present invention. In these situations,the single enantiomers (optically active forms) can be obtained byasymmetric synthesis, synthesis from optically pure precursors, or byresolution of the racemates. Resolution of the racemates can also beaccomplished, for example, by conventional methods such ascrystallization in the presence of a resolving agent, or chromatography,using, for example a chiral HPLC column.

Unless otherwise specified, or clearly indicated by the text, referenceto compounds useful in the combination therapy of the invention includesboth the free base of the compounds, and all pharmaceutically acceptablesalts of the compounds. A preferred salt is the hydrochloride salt.

The term “pharmaceutically acceptable salts” includes derivatives of thedisclosed compounds, wherein the parent compound is modified by makingnon-toxic acid or base addition salts thereof, and further refers topharmaceutically acceptable solvates, including hydrates, of suchcompounds and such salts. Examples of pharmaceutically acceptable saltsinclude, but are not limited to, mineral or organic acid addition saltsof basic residues such as amines; alkali or organic addition salts ofacidic residues such as carboxylic acids; and the like, and combinationscomprising one or more of the foregoing salts. The pharmaceuticallyacceptable salts include non-toxic salts and the quaternary ammoniumsalts of the parent compound formed, for example, from non-toxicinorganic or organic acids. For example, non-toxic acid salts includethose derived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, and nitric; other acceptable inorganicsalts include metal salts such as sodium salt, potassium salt, andcesium salt; and alkaline earth metal salts, such as calcium salt andmagnesium salt; and combinations comprising one or more of the foregoingsalts.

Pharmaceutically acceptable organic salts include salts prepared fromorganic acids such as acetic, trifluoroacetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,HOOC(CH₂)_(n)COOH where n is 0-4; organic amine salts such astriethylamine salt, pyridine salt, picoline salt, ethanolamine salt,triethanolamine salt, dicyclohexylamine salt,N,N′-dibenzylethylenediamine salt; and amino acid salts such asarginate, asparginate, and glutamate, and combinations comprising one ormore of the foregoing salts.

An “effective amount” of a combination of agents is an amount sufficientto provide an observable improvement over the baseline clinicallyobservable signs and symptoms of the disorder treated with thecombination.

The pharmaceutical products can be administrated by oral or other forms,e.g., rectally or by parenteral injection. “Oral dosage form” is meantto include a unit dosage form prescribed or intended for oraladministration. An oral dosage form may or may not comprise a pluralityof subunits such as, for example, microcapsules or microtablets,packaged for administration in a single dose.

The pharmaceutical products can be released in various forms.“Releasable form” is meant to include instant release,immediate-release, controlled-release, and sustained-release forms.

“Instant-release” is meant to include a dosage form designed to ensurerapid dissolution of the active agent by modifying the normal crystalform of the active agent to obtain a more rapid dissolution.

“Immediate-release” is meant to include a conventional or non-modifiedrelease form in which greater than or equal to about 50% or morepreferably about 75% of the active agents is released within two hoursof administration, preferably within one hour of administration.

“Sustained-release” or “extended-release” includes the release of activeagents at such a rate that blood (e.g., plasma) levels are maintainedwithin a therapeutic range but below toxic levels for at least about 8hours, preferably at least about 12 hours, more preferably about 24hours after administration at steady-state. The term “steady-state”means that a plasma level for a given active agent or combination ofactive agents, has been achieved and which is maintained with subsequentdoses of the active agent(s) at a level which is at or above the minimumeffective therapeutic level and is below the minimum toxic plasma levelfor a given active agent(s).

The term “treat”, “treated,” “treating” or “treatment” is used herein tomean to relieve, reduce or alleviate at least one symptom of a diseasein a subject. For example, treatment can be diminishment of one orseveral symptoms of a disorder or complete eradication of a disorder,such as cancer. Within the meaning of the present invention, the term“treat” also denote to arrest, delay the onset (i.e., the period priorto clinical manifestation of a disease) and/or reduce the risk ofdeveloping or worsening a disease. The term “protect” is used herein tomean prevent delay or treat, or all, as appropriate, development orcontinuance or aggravation of a disease in a subject. Within the meaningof the present invention, the disease is associated with a cancer.

The term “subject” or “patient” is intended to include animals, whichare capable of suffering from or afflicted with a cancer or any disorderinvolving, directly or indirectly, a cancer. Examples of subjectsinclude mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats,cats, mice, rabbits, rats, and transgenic non-human animals. In certainembodiments, the subject is a human, e.g., a human suffering from, atrisk of suffering from, or potentially capable of suffering fromcancers.

The term “about” or “approximately” usually means within 20%, morepreferably within 10%, and most preferably still within 5% of a givenvalue or range. Alternatively, especially in biological systems, theterm “about” means within about a log (i.e., an order of magnitude)preferably within a factor of two of a given value.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising, “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein.

As specified above, in one aspect, the instant invention provides a drugcombination useful for treating, preventing, arresting, delaying theonset of and/or reducing the risk of developing, or reversing at leastone symptom of cancer, in a subject comprising administering to thesubject a combination therapy, comprising an effective amount of a RAFinhibitor and a second inhibitor. In some embodiments, the secondinhibitor is a MEK inhibitor. Preferably, these inhibitors areadministered at therapeutically effective dosages which, when combined,provide a beneficial effect. The administration may be simultaneous orsequential.

The term “cancer” is used herein to mean a broad spectrum of tumors,including all solid tumors and hematological malignancies. Examples ofsuch tumors include but are not limited to leukemias, lymphomas,myelomas, carcinomas, metastatic carcinomas, sarcomas, adenomas, nervoussystem cancers and geritourinary cancers. In exemplary embodiments, theforegoing methods are useful in treating adult and pediatric acutelymphoblastic leukemia, acute myeloid leukemia, adrenocorticalcarcinoma, AIDS-related cancers, anal cancer, cancer of the appendix,astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer,bone cancer, osteosarcoma, fibrous histiocytoma, brain cancer, brainstem glioma, cerebellar astrocytoma, malignant glioma, ependymoma,medulloblastoma, supratentorial primitive neuroectodermal tumors,hypothalamic glioma, breast cancer, male breast cancer, bronchialadenomas, Burkitt lymphoma, carcinoid tumor, carcinoma of unknownorigin, central nervous system lymphoma, cerebellar astrocytoma,malignant glioma, cervical cancer, childhood cancers, chroniclymphocytic leukemia, chronic myelogenous leukemia, chronicmyeloproliferative disorders, colorectal cancer, cutaneous T-celllymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewingfamily tumors, extracranial germ cell tumor, extragonadal germ celltumor, extrahepatic bile duct cancer, intraocular melanoma,retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinalstromal tumor, extracranial germ cell tumor, extragonadal germ celltumor, ovarian germ cell tumor, gestational trophoblastic tumor, glioma,hairy cell leukemia, head and neck cancer, hepatocellular cancer,Hodgkin lymphoma, non-Hodgkin lymphoma, hypopharyngeal cancer,hypothalamic and visual pathway glioma, intraocular melanoma, islet celltumors, Kaposi sarcoma, kidney cancer, renal cell cancer, laryngealcancer, lip and oral cavity cancer, small cell lung cancer, non-smallcell lung cancer, primary central nervous system lymphoma, Waldenstrommacroglobulinema, malignant fibrous histiocytoma, medulloblastoma,melanoma, Merkel cell carcinoma, malignant mesothelioma, squamous neckcancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosisfungoides, myelodysplastic syndromes, myeloproliferative disorders,chronic myeloproliferative disorders, nasal cavity and paranasal sinuscancer, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer,ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer,pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorialprimitive neuroectodermal tumors, pituitary cancer, plasma cellneoplasms, pleuropulmonary blastoma, prostate cancer, rectal cancer,rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterinesarcoma, Sezary syndrome, non-melanoma skin cancer, small intestinecancer, squamous cell carcinoma, squamous neck cancer, supratentorialprimitive neuroectodermal tumors, testicular cancer, throat cancer,thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer,trophoblastic tumors, urethral cancer, uterine cancer, uterine sarcoma,vaginal cancer, vulvar cancer, and Wilms tumor.

In particular, the cancer may be associated with a mutation in the B-RAFgene. In some embodiments, the cancer may be a RAF dependent cancer.These cancers include but are not limited to melanoma, breast cancer,colorectal cancers, glioma, lung cancer, ovarian cancer, sarcoma andthyroid cancer.

In a particular embodiment, the therapeutic combination provided hereinis effective for the treatment of moderate to severe cancer in asubject.

Dosages

The optimal dose of the combination of agents for treatment of cancercan be determined empirically for each subject using known methods andwill depend upon a variety of factors, including the activity of theagents; the age, body weight, general health, gender and diet of thesubject; the time and route of administration; and other medications thesubject is taking. Optimal dosages may be established using routinetesting and procedures that are well known in the art.

The amount of combination of agents that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the individual treated and the particular mode of administration.In some embodiments the unit dosage forms containing the combination ofagents as described herein will contain the amounts of each agent of thecombination that are typically administered when the agents areadministered alone.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound that is the lowest dose effective to producea therapeutic effect. Such an effective dose will generally depend uponthe factors described above and is readily determined by one havingskill in the art.

Generally, therapeutically effective doses of the compounds of thisinvention for a patient, when used for the indicated analgesic effects,will range from about 0.0001 to about 1000 mg per kilogram of bodyweight per day, more preferably from about 0.01 to about 50 mg per kgper day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

Pharmaceutical Formulations and Routes of Administration

Provided herein are pharmaceutical formulations comprising a combinationof agents for the treatment of cancer, e.g., melanoma. Thepharmaceutical formulations may additionally comprise a carrier orexcipient, stabilizer, flavoring agent, and/or coloring agent.

Provided herein are pharmaceutical formulations comprising combinationof agents which can be, for example, a combination of two types ofagents: (1) a RAF inhibitor and/or pharmacologically active metabolites,salts, solvates and racemates of the inhibitor and (2) a MEK inhibitorand/or pharmacologically active metabolites, salts, solvates andracemates of the MEK inhibitor.

The combination of agents may be administered using a variety of routesof administration known to those skilled in the art. The combination ofagents may be administered to humans and other animals orally,parenterally, sublingually, by aerosolization or inhalation spray,rectally, intracisternally, intravaginally, intraperitoneally, bucally,or topically in dosage unit formulations containing conventionalnontoxic pharmaceutically acceptable carriers, adjuvants, and vehiclesas desired. Topical administration may also involve the use oftransdermal administration such as transdermal patches or ionophoresisdevices. The term parenteral as used herein includes subcutaneousinjections, intravenous, intramuscular, intrasternal injection, orinfusion techniques.

Methods of formulation are well known in the art and are disclosed, forexample, in Remington: The Science and Practice of Pharmacy, MackPublishing Company, Easton, Pa., 19th Edition (1995). Pharmaceuticalcompositions for use in the present invention can be in the form ofsterile, non-pyrogenic liquid solutions or suspensions, coated capsules,suppositories, lyophilized powders, transdermal patches or other formsknown in the art.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3 propanediol or 1,3butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P. and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose any blandfixed oil may be employed including synthetic mono or di glycerides. Inaddition, fatty acids such as oleic acid find use in the preparation ofinjectables. The injectable formulations can be sterilized, for example,by filtration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform may be accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such as polylactidepolyglycolide. Depending upon the ratio of drug to polymer and thenature of the particular polymer employed, the rate of drug release canbe controlled. Examples of other biodegradable polymers includepoly(orthoesters) and poly(anhydrides). Depot injectable formulationsmay also be prepared by entrapping the drug in liposomes ormicroemulsions, which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,acetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, EtOAc, benzylalcohol, benzyl benzoate, propylene glycol, 1,3 butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, the oral compositions can alsoinclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring, and perfuming agents.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulations, ear drops, and the like are also contemplatedas being within the scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Compositions of the invention may also be formulated for delivery as aliquid aerosol or inhalable dry powder. Liquid aerosol formulations maybe nebulized predominantly into particle sizes that can be delivered tothe terminal and respiratory bronchioles.

Aerosolized formulations of the invention may be delivered using anaerosol forming device, such as a jet, vibrating porous plate orultrasonic nebulizer, preferably selected to allow the formation of anaerosol particles having with a mass medium average diameterpredominantly between 1 to 5 μm. Further, the formulation preferably hasbalanced osmolarity ionic strength and chloride concentration, and thesmallest aerosolizable volume able to deliver effective dose of thecompounds of the invention to the site of the infection. Additionally,the aerosolized formulation preferably does not impair negatively thefunctionality of the airways and does not cause undesirable sideeffects.

Aerosolization devices suitable for administration of aerosolformulations of the invention include, for example, jet, vibratingporous plate, ultrasonic nebulizers and energized dry powder inhalers,that are able to nebulize the formulation of the invention into aerosolparticle size predominantly in the size range from 1 to 5 μm.Predominantly in this application means that at least 70% but preferablymore than 90% of all generated aerosol particles are within 1 to 5 μmrange. A jet nebulizer works by air pressure to break a liquid solutioninto aerosol droplets. Vibrating porous plate nebulizers work by using asonic vacuum produced by a rapidly vibrating porous plate to extrude asolvent droplet through a porous plate. An ultrasonic nebulizer works bya piezoelectric crystal that shears a liquid into small aerosoldroplets. A variety of suitable devices are available, including, forexample, AERONEB and AERODOSE vibrating porous plate nebulizers(AeroGen, Inc., Sunnyvale, Calif.), SIDESTREAM nebulizers (Medic AidLtd., West Sussex, England), PARI LC and PARI LC STAR jet nebulizers(Pari Respiratory Equipment, Inc., Richmond, Va.), and AEROSONIC(DeVilbiss Medizinische Produkte (Deutschland) GmbH, Heiden, Germany)and ULTRAAIRE (Omron Healthcare, Inc., Vernon Hills, Ill.) ultrasonicnebulizers.

Compounds of the invention may also be formulated for use as topicalpowders and sprays that can contain, in addition to the compounds ofthis invention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel. The compounds of the present invention can also be administeredin the form of liposomes. As is known in the art, liposomes aregenerally derived from phospholipids or other lipid substances.Liposomes are formed by mono or multi lamellar hydrated liquid crystalsthat are dispersed in an aqueous medium. Any nontoxic, physiologicallyacceptable and metabolizable lipid capable of forming liposomes can beused. The present compositions in liposome form can contain, in additionto a compound of the present invention, stabilizers, preservatives,excipients, and the like. The preferred lipids are the phospholipids andphosphatidyl cholines (lecithins), both natural and synthetic. Methodsto form liposomes are known in the art. See, for example, Prescott(ed.), “Methods in Cell Biology,” Volume XIV, Academic Press, New York,1976, p. 33 et seq.

EXAMPLES Example 1: Identification and Characterization of CRAFResistance Mutations In Vitro

To identify somatic RAF mutations that confer resistance to RAFinhibitors, saturating random mutagenesis screens were performed usingthe XL1-Red bacterial system and retroviral vectors expressing C-RAFcDNAs (Emery et al., Id. 2009, Wagle et al., Id. 2011). The resultingrandomly mutagenized saturating cDNA library of CRAF mutations wasexpressed in A375 melanoma cells harboring the BRAF^(V600E) mutationwhich is highly responsive to the RAF inhibitors (Li et al., 2007; Tsaiet al., 2008, Emery et al., Id. 2009). These cells were cultured for 4weeks in the presence of fully inhibitory concentrations of PLX4720 (1.5μM) and the resistant clones emerged were pooled and characterized bymassive parallel sequencing (Emery et al., Id. 2009, Wagle et al., Id.2011). All C-RAF mutations examined (FIG. 1A) could be stably expressedin A375 cells. (FIG. 9A). Furthermore, 8 of the 10 most prominent C-RAFmutations conferred biochemical resistance to the RAF inhibitor PLX4720in A375 cells, as evidenced by p-MEK and p-ERK levels (FIG. 9B). One ofthese alleles (CRAF^(G361A)) conferred substantial “paradoxical”activation of p-MEK at high PLX4720 concentrations (FIG. 9B).

The CRAF mutations attained through the screen were mapped on to thecrystal structure of the CRAF kinase domain (340-618) (Hatzivassiliou etal., 2010) (PDB code: 30MV) (FIG. 1B). Three of the mutations were notmapped since the full length structure of CRAF has not been solved todate. The CRAF resistance alleles clustered towards two distinctregions, outside or within the regulatory region (N-terminus) and in thekinase domain (C-terminus) (FIG. 1C). CRAF consists of two 14-3-3consensus binding sites and two of the resistance alleles S257P andP261T occupied one of the 14-3-3 consensus binding sites in the CR2except the E104K allele in CR1 region (FIG. 1B).

The second category of resistance alleles encompasses the kinase domainincluding the glycine rich loop. Mutations such as G356E and G361A werefound in the glycine residue of the ATP-binding P-loop, GxGxxG motif.The P-loop mutations has been found in several protein kinases(Christopher et al., 2007) including BRAF which possess cellulartransformation capacity by activating Mek (Wan et al., Cell 116:855-867, 2004; Garnett et al., Id. 2005). The other subset of mutations(S427T, D447N, M4691, E478K and R554K) populated outside the activationsegment (FIG. 1B).

Example 2: Functional Characterization of C-RAF Resistance Mutants

To study the functional consequences of the identified C-RAF resistancealleles, the representative mutations (FIG. 1A) were introduced into andexpressed in A375 melanoma cells. Biochemical analysis followed bytreatment with Raf inhibitor, PLX4720 attenuated Mek phosphorylation(pMek) and Erk phosphorylation (pErk) at a concentration of 2 μM (FIG.2A) in A375 cells and WT-C-RAF expressing cells. However, the resistancealleles showed sustained pMek and pErk at the same (FIG. 2A)concentration. Also, the resistance alleles clustered towards the 14-3-3binding region (S257P and P261T) and one in the ATP binding region,particularly G361A conferred profound pharmacological resistance toPLX4720. These mutants increased the PLX4720 GI₅₀ values by 100 fold(S257P and P261T) and 30 fold (G361A) when compared to the WT (0.4 μM)(FIG. 2C). Moreover, stability of C-Raf has been correlated to S259 andS621 phosphorylation (FIG. 1C) and subsequent 14-3-3 binding andactivation of C-RAF has been correlated with phosphorylation of S338 andS621 residues (FIG. 1C) (Wellbrock et al., Id. 2004). PLX4720 inducedthe phosphorylation of S338 and a modest increase of S621 particularlyin S257P, P261T and G361A resistance mutants which is consistent withparadoxical activation of pMek in these variants (FIG. 2B). This C-RAFactivation of MEK/ERK signaling was suppressed by pharmacologic MEKinhibition (FIG. 2G). The WT expressing cells exhibited a modestincrease in S338 but not in S621. On the contrary, the S259 site wasphosphorylated under basal conditions and ectopic expression of WT-C-RAFexacerbated the effect in the absence of PLX4720 (FIG. 2B). However,PLX4720 also induced S259 phosphorylation in the resistance mutants butcomparatively these mutants exhibited lower S259 phosphorylation levels(FIG. 2B), but this effect was attenuated in the presence of AZD6244with diminishing levels of pErk (FIG. 2E). As C-RAF is highly modulatedand activated by phosphorylation, these data suggest that there is afeedback activation and inhibition loop which is constantly at workmaintaining robustness and stringency to the MAPK signaling output.Hence, the C-RAF resistance allele's activity might be modulated by abalance between the amount of phosphorylation attained by sites whichrender activity (S338, S621) and sites that render inhibition (S259).Only one allele (G361A) conferred evidence of pharmacologic resistanceto MEK inhibition (FIGS. 2F and 2G).

Example 3: C-RAF Resistance Mutants Exhibit Increased Association withB-RAF

The cumulative data above show that the resistance alleles encompassingthe 14-3-3 consensus binding site (S257P and P261T) and the ATP bindingregion (G361A) (FIG. 1C) confer resistance due to decreased inhibitionof phosphorylated MEK and ERK. Also, it has been shown that a doublemutant of S259/S621A completely abrogates the interaction between C-RAFand 14-3-3 without affecting the interaction with RAS or MEK (Tzivion etal., Nature 394: 88-92, 1998). To investigate the underlying mechanism,we immunoprecipitated C-RAF from 293/T cells ectopically expressing allthe resistance alleles identified during the initial screen. Mutationsthat decreased interaction with 14-3-3 and increased the interactionwith B-RAF maintained a higher MEK and ERK phosphorylation status whencompared to the WT (FIG. 3A) and also increased C-RAF kinase activity invitro (data not shown). These results corroborated the interactionstatus of 14-3-3 and B-RAF in A375 cells expressing C-RAF alleles (FIG.3B). Hence, these resistance mutants possess higher activity towards itssubstrate even in the absence of an oncogenic driver such as Ras (Weberet al., 2001).

Example 4

Biochemical Characterization of C-RAF Resistance alleles using(S)-methyl1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate.

To exclude the possibility of inefficient binding of PLX4720 to theresistance mutants, mutant C-RAF responses to (S)-methyl1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate(Novartis), in A375 cells and cells expressing WT-C-RAF(FIG. 3C) wereexamined. The C-RAF resistance alleles (S257P, P261T) increased(S)-methyl1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamateG150 values by ˜10,000 and ˜30,000 fold respectively and G361A by 20fold (FIG. 3C). As observed in FIGS. 2C and E, the response to PLX4720and AZD6244 remained same (FIGS. 3B and D). Furthermore, biochemicallythe C-RAF resistance mutants conferred resistance to (S)-methyl1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamateeven at a concentration of 1 μM (FIG. 3A).

Example 5: C-RAF Resistance Mutants Confer Resistance to Vemurafenib(PLX4032)

Vemurafenib is highly responsive to BRAF^(V600E) mutations but causes aparadoxical activation of Mek and Erk in cells expressing oncogenic Ras(Hatzivassiliou et al., 2010; Heidorn et al., 2010; Poulikakos et al.,2010). C-RAF resistance alleles were tested to determine whether theC-RAF resistance alleles exhibited similar response to Vemurafenib inthe presence of oncogenic B-RAF. A375 cells expressing VVT-C-RAF (0.4μM) (FIG. 5A) showed comparable GI₅₀ values to that of PLX4720 treatedVVT-C-RAF cells (FIG. 2C). The resistance conferred by G361A was 50 foldhigher than the WT, whereas surprisingly, the S257P and P261T displayedan increased resistance towards Vemurafenib. As, these mutants conferredresistance even in the absence of oncogenic Ras, it was furtherdetermined, if resistance to the Raf inhibitor was due to an increasedactivity. Total C-RAF was immunoprecipitated from extracts of A375 cellsexpressing the C-RAF variants in the presence and absence ofVemurafenib. Mutations that displayed high resistance (S257P and P261T)during pharmacological inhibition (FIG. 5A) had increased kinaseactivity in vitro compared to the WT (FIG. 5B); however G361A resistancemutant displayed even higher kinase activity in presence of the drug(FIG. 5B). Moreover, it is shown that C-RAF resistance alleles remainsensitive to combinatorial treatment with PLX4720 and AZD6244 (FIGS. 5C,D and E).

Together, these data are consistent with the notion that just an optimalamount of Erk signaling is required for the cells to confer resistance.

Example 6: C-RAF Resistance Alleles Enable Paradoxical C-RAF Activationand Enhanced Dimerization

Paradoxical MEKIERK activation induced by RAF inhibitors involvesdimerization of RAF proteins (Hatzivassiliou et al., 2010; Poulikakos etal., 2010). Moreover, a truncated form of BRAF^(V600E) that showsenhanced dimerization confers resistance to RAF inhibitors (Poulikakoset al., 2011). To determine whether the C-RAF resistance mutationsmediate resistance through increased dimerization, co-transfections wereperformed in 293/T cells using expression constructs in which severalrepresentative C-RAF resistance alleles were differentially tagged withtwo distinct epitopes (His/V5 or Flag). Immunoprecipitation reactionswere carried out using Ni²⁺ beads (to capture the His-tagged protein)followed by immunoblotting using anti-Flag antibody. In theseexperiments, all C-RAF mutations that conferred pharmacologic resistanceto RAF inhibitors also exhibited increased homodimerization compared towild type C-RAF (S257P, P261T, G361A, and E478K). As expected, theincreased dimerization generally correlated with increased p-MEK levels(FIG. 6A, input lysate). Similar results were observed whenHis/V5-tagged C-RAF mutants were co-transfected with Flag-taggedwild-type C-RAF (FIG. 10A), although the magnitude of MEKIERK activationseemed qualitatively reduced (FIG. 10A, input lysate). The three C-RAFmutants that conferred the most profound pharmacologic resistance toPLX4720 and vemurafenib (S257P, P261T, and G361A) (FIGS. 2C and 2F) alsoshowed evidence of increased total protein accumulation (FIG. 6A, inputlysate). Thus, the resistance phenotype linked to C-RAF mutationscorrelated strongly with RAF dimerization.

In 293T cells (which lack oncogenic BRAF mutations), the increased C-RAFdimerization engendered by the presence of resistance mutations wassustained but not further enhanced upon exposure of transfected cells toRAF inhibitor (vemurafenib; FIG. 6B). However, both C-RAF activation(evidenced by S338 phosphorylation) and downstream MEKIERK signalingwere robustly induced by the RAF inhibitor (FIG. 6B, input lysate). Todetermine the effects of these resistance mutations on intrinsic C-RAFkinase activity, in vitro kinase reactions were performed from 293Tcells cultured in the presence or absence of RAF inhibitor. In theabsence of drug, steady-state C-RAF kinase activity (p-MEK) was notmeasurably increased by the resistance mutations in most cases (FIG.6C). CRAF^(G361A) was the one exception to this; here, modeststeady-state kinase activity was detected that correlated with robustintrinsic p-MEK levels in the corresponding whole cell lysates (FIG.6C). In contrast, treatment of 293T cells with 2 μM vemurafenib prior tothe in vitro kinase assays resulted in a marked up-regulation of C-RAFkinase activity in all resistance alleles examined (FIG. 6C). Similarexperiments in BRAF^(V600E) melanoma cells (A375) revealed an increasein intrinsic kinase activity in the three most potent C-RAF resistancemutants examined (S257P, P261T, and G361A; FIG. 6D). This kinaseactivity was further augmented upon exposure of these cells tovemurafenib (FIG. 6D), as observed in 293T cells. Together, theseresults suggested that potent C-RAF resistance mutants enhanced both RAFdimerization and RAF inhibitor-mediated C-RAF kinase activity.

Example 7:C-RAF Resistance Mutants Exhibit Reduced 14-3-3 Binding andIncreased B-RAF Heterodimerization

The cumulative data above suggested that C-RAF mutations encompassingits 14-3-3 consensus binding site (S257P and P261T) and the ATP bindingregion of the P loop (G361A) conferred pharmacological and biochemicalresistance to RAF inhibition, enhanced RAF dimerization, and increasedC-RAF kinase activation upon treatment with RAF inhibitors. Toinvestigate the role of 14-3-3 protein binding in relation to RAFdimerization, immunoprecipitation experiments were performed from cellsengineered to ectopically express C-RAF resistance alleles. To examinethe effects of pharmacologic RAF inhibition on 14-3-3 binding and B-RAFheterodimerization, these experiments were conducted in both the absenceand presence of RAF inhibition (in this case, vemurafenib). In theabsence of RAF inhibitor, the C-RAF resistance alleles S257P, P261T andG361A tended to demonstrate reduced interactions with 14-3-3 ζ andincreased interactions with B-RAF in both 293T cells (FIG. 7A) and, inparticular, A375 (BRAF^(V600E)) melanoma cells (FIG. 7B). In 293T cells,the enhanced C-RAF/B-RAF heterodimerization triggered by C-RAF mutationscorrelated with C-RAF protein stabilization and robust MEKIERKphosphorylation (FIG. 7A). On the other hand, the robust MEK/ERKactivation observed in BRAF^(V600E) melanoma cells was only marginallyenhanced by the C-RAF resistance mutants (FIG. 7B); this result wasexpected given the constitutive oncogenic B-RAF signaling in thesecells. Interestingly, one of the C-RAF mutants (G356E) exhibited verylow 14-3-3ζ binding in both cellular contexts (FIGS. 7A and 7B);however, C-RAF^(G356E) showed no enrichment in B-RAF heterodimerizationand no increase in MEKIERK signaling under steady-state conditions.These results suggest that while reduced 14-3-3 binding may promoteenhanced mutant C-RAF dimerization, some degree of 14-3-3 binding(perhaps within the C-terminal domain) is needed to promote maximalRAF-dependent signaling.

As expected, the RAF inhibitor vemurafenib induced B-RAF/C-RAFheterodimerization in 293/T cells ectopically expressing wild-type C-RAF(FIG. 7A), but abrogated this heterodimerization in A375 melanoma cells(FIG. 7B). In contrast, ectopic expression of the most robust C-RAFresistance mutants enabled sustained B-RAF heterodimerization even inthe presence of drug in A375 cells (FIG. 7B). These results suggest thatBRAF^(V600E) assumes a dominant conformation that favorsheterodimerization with resistance-associated C-RAF variants.Pharmacologic RAF inhibition had variable effects on the 14-3-3/C-RAFinteraction depending on the cellular genetic context. In A375 cells(BRAF^(V600E)), vemurafenib modestly decreased 14-3-3ζ binding towild-type C-RAF, but had no effect in the setting of the C-RAF^(S257P),C-RAF^(P261T) and C-RAF^(G361A) mutants (FIG. 7B). On the other hand,vemurafenib enhanced these 14-3-3/C-RAF interactions in 293/T cells(FIG. 7A). These findings lent further support to the premise that theresistance phenotype conferred by these C-RAF mutants in theBRAF^(V600E) context involved enhanced RAF dimerization, whichcorrelated with diminished 14-3-3/C-RAF interactions.

Example 8: Enhanced MEKIERK Signaling by C-RAF Resistance MutantsRequires Dimerization

To test whether C-RAF dimerization is necessary for the enhanced MEKIERKsignaling conferred by C-RAF resistance mutants, an arginine-histidinemutation was introduced at residue R401 (C-RAF^(R401H)) (FIG. 10B). Thismutant has previously been shown to disrupt C-RAF homodimerization((Hatzivassiliou et al., Nature 464: 431-435, 2010; Poulikakos et al.,Nature 464:427-430, 2010). The R401H dimerization deficient mutation wasintroduced into the respective C-RAF resistance alleles. As expected,C-RAF double mutants were rendered largely incapable of enhanced MEKIERKsignaling (FIG. 8A). Next, co-transfections were performed usingdifferentially epitope-tagged C-RAF resistance/R401H double mutants. Asdescribed earlier, the C-RAF resistance alleles augmented C-RAFhomodimerization in a manner unaffected by RAF inhibitor (FIG. 8B). Incontrast, introduction of the R401H allele suppressed C-RAFhomodimerization and abrogated MEKIERK signaling in most C-RAF mutantcontexts examined. The exception to this was the C-RAF^(G361A) allele,which exhibited constitutive (albeit markedly reduced) MEKIERKactivation that was further induced by vemurafenib exposure, even whenco-expressed with the dimerization-deficient double mutant. Togetherwith the in vitro kinase activity results above, these data suggest thatthe C-RAF^(G361A/R401H) allele may also contain increased intrinsickinase activity. Overall, these results provide direct evidence that theenhanced MEK/ERK signaling conferred by C-RAF resistance mutantsrequires RAF dimerization. They may also provide a rationale for thefuture development of allosteric RAF inhibitors that disrupt the RAFdimerization interface.

Methods Cell Culture

293/T cells, A375 cells (ATCC) and Phoenix cells (Allele Biotech) werecultured and maintained at 37° C. in Dulbecco's modified Eagle's mediumsupplemented with 10% fetal bovine serum in a humidified atmospherecontaining 5% CO₂.

C-RAF Random Mutagenesis Screen

C-RAF cDNA was cloned into pWZL-Blast vector (gift from J. Boehm and W.C. Hahn) by recombinational cloning (Invitrogen). Specific mutationswere introduced into c-raf cDNA using QuickChange II Site DirectedMutagenesis (Stratagene). Random mutagenesis was done based onestablished protocol (Emery et al., Id. 2009). The mutagenized C-RAFplasmid was used to infect A375 melanoma cells. Following selection withBlasticidin, cells were plated on 15-cm dishes and cultured in thepresence of RAF inhibitor, PLX4720 (1.5 μM) for 4 weeks until resistantclones emerged.

Sequencing of c-RAF DNA

PLX4720 resistant cells emerging from the random mutagenesis screenswere pooled and genomic DNA was prepared (Qiagen DNeasy). C-RAF cDNA wasamplified from genomic DNA using primers specific to flanking vectorsequence at the 5′ and 3′ end and sequenced by the Sanger method usingestablished protocols.

Analysis of Massively Parallel Sequencing

Raw data from massively parallel sequencing lanes (Illumina; 2-3 million36-base-pair sequences per lane) were analyzed using a “next-generation”sequencing analysis pipeline (Emery et al., PNAS, 2009.). Output fromdata files representing the nucleotide sequence, per-base qualitymeasure, variants detected, and alignment to cDNA reference sequence (asdetermined by alignment with the ELAND algorithm) were integrated andprocessed for each run. Coverage (i.e., the number of fragmentsincluding each base of the cDNA reference) was determined for all bases,and variant alleles were mapped from individual DNA fragments onto thereference sequence. The frequency of variation for each nonwild-typeallele was determined, and an average variant score (AVS) was calculatedas the mean of all quality scores for the position and variant allele inquestion. All coding mutations were translated to determine the aminoacid variation (if any) and data for high-frequency (>0.5%) andhigh-quality (AVS>7) mutations were loaded into the CCGD resultsdatabase.

Retroviral Infections

Phoenix cells (70% confluent) were transfected with pWZLBlast-C-RAF orthe mutants using Fugene 6 (Roche). Supernatants containing virus werepassed through a 0.45-μm syringe. The A375 cells were infected for 16 hwith virus together with polybrene (4 μg/mL, Sigma). The selectivemarker blasticidin (3 μg/mL) was introduced 48 h postinfection.

Western Blot Analysis

Samples were extracted after washing twice with PBS and lysed with 150mM NaCl, 50 mM Tris pH 7.5, 1 mM EDTA, PMSF, Sodium Fluoride, SodiumOrthovanadate and protease inhibitor cocktail in the presence of 1%NP-40. The protein content was estimated with protein assay reagent(Bio-Rad) according to the manufacturer's instructions. Equal amounts ofwhole cell lysates were loaded onto and separated by 8-16% SDS-PAGEready-made gels. Proteins were transferred to polyvinylidene difluoridemembranes in a Trans-Blot apparatus. Membranes were blocked with 5% skimmilk in TBS containing 0.1% Tween 20 for 1 h at room temperature orovernight at 4° C. Membranes were then incubated with monoclonal orpolyclonal antibody raised against the protein of interest for 1 h atroom temperature or overnight at 4° C. followed by three washes with TBScontaining 0.1% Tween 20. The immunoreactivity of the primary antibodiesC-RAF, S259C-RAF, S338C-RAF, S621C-RAF, pERK, ERK, pMEK, MEK, 14-3-3 ζFlag (Cell Signaling), B-RAF (Santa Cruz Biotechnology) and actin(Sigma) was visualized with a secondary anti-rabbit (BD TransductionLaboratories) or anti-mouse (Santa Cruz Biotechnology) antibodiesconjugated with horseradish peroxidase and subsequent development withECL Plus (Amersham Biosciences) and autoradiography on X-OMAR TAR films.The bands were scanned and quantified by the Gel Doc system using theQuantity One software.

Immunoprecipitation

For immunoprecipitation with C-RAF antibody (BD Biosciences) proteinG-Sepharose slurry (Thermo Scientific) was washed with 1×PBS andincubated with C-RAF antibody (BD Biosciences) or normal mouse IgG(control) for 1 h at 4° C. After three washes with lysis buffer, thebeads were incubated with whole cell lysates (0.5 mg of total protein)for 2 h and then washed three times with lysis buffer. The proteins werethen eluted by boiling in 1×SDS-sample buffer.

C-RAF Kinase Assay

The 293T cells (70% confluent) were transfected with 6 μg pc-DNA withHis or V5 tag towards the C-terminal containing C-RAF-WT and C-RAFvariant alleles. Cells were treated with vemurafenib (Allele Biotech)for 1h and 48h post-transfection, lysates were extracted by generalprotocol. Immunoprecipitation using cobalt beads was performed overnightfor 1 hr at 4° C. The protein-bound cobalt beads where incubated with 20μL ATP/magnesium mixture (20 mM Mops pH 7.2, 25 mM β-glycerophosphate, 5mM EGTA, 1 mM Na3VO4, 1 mM DTT, 75 mM MgCl2, and 0.5 mMATP), 20 μL ofdilution buffer (20 mM Mops, pH 7.2, 25 mM-glycerol phosphate, 5 mMEGTA, 1 mM sodium orthovanadate, 1 mM DTT), and 1 μg of inactive MEK(obtained from Millipore) for 30 min at 30° C. The phosphorylated MEKproduct was detected by immunostaining using a p-MEK antibody (CellSignaling Technology), and relative p-MEK signals were quantified usingdensitometry, normalized to the amount of input C-RAF, and compared toC-RAF-VVT as a reference.

C-RAF Kinase Assay (A375)

A375 cells infected with VVT and mutant C-RAF alleles were cultured inthe absence and presence of PLX4032 (Allele Biotech) for 16 h. Lysateswere prepared with 150 mM NaCl, 50 mM Tris pH 7.5, 1 mM EDTA, PMSF,Sodium Fluoride, Sodium Orthovanadate and protease inhibitor cocktail inthe presence of 1% NP-40. Immunoprecipitation with C-RAF antibody wasperformed overnight and bound beads were washed three times with lysisbuffer, followed by kinase buffer (1×). The beads were incubated with 20ul of ATP/Magnesium mixture (Millipore) and 0.5 μg of inactive MEK(Millipore) for 30 min at 30° C. The phosphorylated substrate MEK wasdetected by immunoblotting.

Pharmacologic Growth Inhibition Assays

Cultured cells were seeded into 96-well plates at a density of 3,000cells per well for all melanoma short-term cultures including A375.After 16 h, serial dilutions of the compound were performed in DMSO andtransferred to cells to yield drug concentrations based on the potencyof the drug, ensuring that the final volume of DMSO did not exceed 1%.The B-RAF inhibitor PLX4720 (purchased from Symansis, PLX4032 (purchasedfrom Allele Biotech), AZD6244 (purchased from Selleck Chemicals) andGSK1120212 (Ourchased from Active Biochem). Following addition of thedrug, cell viability was measured using the Cell-Titer-96 aqueousnon-radioactive proliferation assay (Promega) after 4 days. Viabilitywas calculated as a percentage of the control (untreated cells) afterbackground subtraction. A minimum of six replicates was made for eachcell line and the entire experiment was repeated at least three times.The data from the pharmacologic growth-inhibition assays were modeledusing a nonlinear regression curve fit with a sigmoidal dose-response.These curves were displayed using GraphPad Prism 5 for Windows(GraphPad). GI50 values were calculated by determining the slope of theline connecting the data points that flanked the 50% point.

The definitions and disclosures provided herein govern and supersede allothers incorporated by reference. Although the invention herein has beendescribed in connection with preferred embodiments thereof, it will beappreciated by those skilled in the art that additions, modifications,substitutions, and deletions not specifically described may be madewithout departing from the spirit and scope of the invention as definedin the appended claims. It is therefore intended that the foregoingdetailed description be regarded as illustrative rather than limiting,and that it be understood that it is the following claims, including allequivalents, that are intended to define the spirit and scope of thisinvention.

1. An expression vector comprising an isolated nucleic acid moleculeencoding a mutant C-RAF polypeptide having C-RAF activity, wherein saidmutant C-RAF polypeptide comprises at least one amino acid substitutionas compared to a wild type C-RAF polypeptide comprising SEQ. ID. NO. 2,the at least one amino acid substitution conferring resistance to one ormore RAF inhibitors on a cell expressing the mutant C-RAF polypeptide.2. A host cell comprising the expression vector of claim
 1. 3. Themutant C-RAF polypeptide according to claim 1, wherein the at least oneamino acid substitution occurs at one or more amino acid positionsselected from the group consisting of 104E, 257S, 261P, 356G, 361G,427S, 447D, 469M, 478E and 554R.
 4. The mutant C-RAF polypeptideaccording to claim 1, wherein the at least one substitution is selectedfrom the group consisting of 104E>K, 257S>P, 261P>T, 356G>E, 361G>A,427S>T, 447D>N, 469M>I, 478E>K and 554R>K.
 5. The mutant C-RAFpolypeptide according to claim 1, wherein the at least one substitutionis selected from the group consisting of 257S, 261P and 361G.
 6. Themutant C-RAF polypeptide according to claim 1, wherein the RAF inhibitoris selected from the group consisting of RAF265, sorafenib, SB590885,PLX 4720, PLX4032, GDC-0879, ZM 336372 and (S)-methyl1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate.7. The mutant C-RAF polypeptide according to claim 1, wherein the RAFinhibitor is PLX4032.
 8. An antibody preparation which specificallybinds to a polypeptide encoded by the expression vector of claim
 1. 9. Amethod of treating a subject having cancer, the method comprising: (a)extracting nucleic acid from cells of a cancer of the patient; (b)assaying at least a portion of a nucleic acid molecule encoding a C-RAFpolypeptide for the presence of one or more mutations in a nucleic acidmolecule encoding a C-RAF polypeptide that alter the identity of anamino acid residue at one or more amino acids of the encoded C-RAFpolypeptide as compared to a wild type C-RAF polypeptide at one or morepositions selected from the group consisting of 104E, 257S, 261P, 356G,361G, 427S, 447D, 469M, 478E and 554R; and (c) administering aneffective amount of a RAF inhibitor and an effective amount of a secondinhibitor to the subject when the nucleic acid molecule includesnucleotides that alter the amino acid reside at one or more amino acidsof the encoded C-RAF polypeptide as compared to a wild type C-RAFpolypeptide.
 10. The method according to claim 9, wherein the secondinhibitor is a MEK inhibitor.
 11. The method according to claim 9,wherein the RAF inhibitor is selected from the group consisting ofRAF265, sorafenib, SB590885, PLX 4720, PLX4032, GDC-0879, ZM 336372 and(S)-methyl1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate.12. The method according to claim 9, wherein the MEK inhibitor isselected from the group consisting of CI-1040/PD184352, AZD6244,PD318088, PD98059, PD334581, RDEA119,6-Methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)-quinoline-3-carbonitrileand4-[3-Chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)-phenylamino]-6-methoxy-7-(3-morpholin-4-yl-propoxy)-quinoline-3-carbonitrile.13. The method according to claim 9, wherein the cancer is selected fromthe group consisting of melanoma, breast cancer, colorectal cancers,glioma, lung cancer, ovarian cancer, sarcoma and thyroid cancer.
 14. Themethod according to claim 9, wherein the cancer is a RAF dependentcancer.
 15. The method according to claim 9, wherein the cancer ismelanoma.
 16. A method of identifying a subject having cancer who islikely to benefit from treatment with a combination therapy with a RAFinhibitor and a second inhibitor, the method comprising: (a) extractingnucleic acid from cells of a cancer of the patient; and (b) assaying atleast a portion of a nucleic acid molecule encoding a C-RAF polypeptide;wherein the presence of one or more nucleotides that alter the identityof an amino acid residue at one or more amino acids of the encodedmutant C-RAF polypeptide relative to the amino acid at one or morepositions of the wild type C-RAF polypeptide at one or more of aminoacid positions selected from the group consisting of 104E, 257S, 261P,356G, 361G, 427S, 447D, 469M, 478E and 554R indicates a need to treatthe subject with a RAF inhibitor and a second inhibitor.
 17. The methodaccording to claim 16, further comprising administering a RAF inhibitorand a second inhibitor to the subject.
 18. The method according to claim16, wherein the presence of one or more nucleotides that alter theidentity of an amino acid residue at one or more amino acids of theencoded mutant C-RAF polypeptide occurs at one or more amino acidpositions selected from the group consisting of 104E>K, 257S>P, 261P>T,356G>E, 361G>A, 427S>T, 447D>N, 469M>, 478E>K and 554R>K.
 19. The methodaccording to any one of claim 16, wherein the presence of one or morenucleotides that alter the identity of an amino acid residue at one ormore amino acids of the encoded mutant C-RAF polypeptide occurs at oneor more amino acid positions selected from the group consisting of 257S,261P and 361G.
 20. The method according to claim 16, wherein the secondinhibitor is a MEK inhibitor.